The drug inhibits neutrophil production of reactive oxygen species (ROS) induced by various agonists (PMA, fMLP, opsonized yeast). It does not act as a direct radical scavenger and has no effect on NADPH oxidase assembly or activity in a cell-free system. The inhibition likely occurs downstream of protein kinase C (PKC) in the signal transduction pathway. [2]

B220 is an antiviral drug that prevents human cytomegalovirus (CMV), HSV-1, and HSV-2 from proliferating [1]. B220 suppresses the generation of intracellular reactive oxygen species as well as the release of reactive oxygen species from neutrophils. B220 does not directly scavenge oxygen radicals, nor does it directly affect the activity of the assembled oxidase to provide this inhibition. B220 inhibits neutrophils' capacity to engulf fluorescein-labeled opsonized yeast cells in their phagocytosis. Compared to control cells, cells that were preincubated with B220 (10 µg/mL) and subsequently activated with the formylated peptide fMLP had less C3 receptors on their surface [2].

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B-220 (5 or 10 μg/mL) inhibited extracellular release of superoxide anions from human neutrophils stimulated with the chemotactic peptide fMLP, as measured by isoluminol-enhanced chemiluminescence. A dose-dependent inhibition was observed. [2]
B-220 (5 or 10 μg/mL) inhibited extracellular superoxide release from human neutrophils stimulated with PMA (PKC activator). [2]
B-220 (5 or 10 μg/mL) inhibited intracellular production of reactive oxygen species in human neutrophils stimulated with PMA or opsonized yeast particles, measured by luminol-enhanced chemiluminescence in the presence of SOD and catalase. [2]
B-220 (10 μg/mL) showed no direct superoxide anion scavenger activity in a cell-free xanthine/xanthine oxidase system, as determined by SOD-inhibitable cytochrome c reduction. [2]
B-220 (10 μg/mL) showed no hydrogen peroxide scavenger activity in a cell-free HRP-PHPA system. [2]
B-220 (10 μg/mL) had no effect on superoxide production in a cell-free NADPH oxidase system using subcellular fractions (specific granules as membrane source and cytosol S2) from disintegrated neutrophils, activated with SDS and GTPγS. [2]
B-220 (10 μg/mL) reduced phagocytosis of serum-opsonized FITC-labeled yeast cells by human neutrophils by approximately 20% (determined by flow cytometry). [2]
B-220 (10 μg/mL) reduced fMLP-induced mobilization of CR3 (complement receptor 3) to the neutrophil cell surface, measured by phycoerythrin-conjugated anti-CD11b antibody binding and flow cytometry. [2]
B-220 (10 μg/mL) reduced fMLP-induced secretion of vitamin B12-binding protein (a specific granule marker) from human neutrophils by approximately 20%, measured using the cyanocobalamin technique. [2]

An ointment containing 1% by weight B-220 in white petrolatum was administered topically to 10 human volunteers suffering from recurrent herpes labialis. Administration was performed repeatedly every day by applying a thin layer to affected areas. All subjects reported that pain disappeared or was substantially alleviated within 1-2 days, and redness disappeared or was substantially reduced within 2-4 days. [1]

A cell-free superoxide anion generating system was used to test scavenger activity. The reaction mixture contained xanthine, cytochrome c, and catalase. Generation of superoxide was initiated by adding xanthine oxidase. The rate of superoxide production was recorded as the SOD-inhibitable increase in absorbance at 550 nm. B-220 (10 μg/mL) was added to the cuvette, and no significant difference in superoxide production was observed compared to control, indicating no scavenger activity for superoxide anions. [2]
A cell-free hydrogen peroxide (H2O2) system was used to test scavenger activity. The cuvette contained PHPA and H2O2. HRP was added to induce oxidation of PHPA, and the increase in fluorescence of oxidized PHPA (excitation 317 nm, emission 400 nm) was proportional to the amount of H2O2 present. B-220 (10 μg/mL) was added, and fluorescence was measured at different time points (1, 2, 3, and 5 min) after HRP addition. No significant difference in fluorescence increase was observed, indicating no scavenger activity for H2O2. [2]
A cell-free NADPH oxidase system was used to assess effects on oxidase assembly and activity. Subcellular fractions from disintegrated human neutrophils were prepared by nitrogen cavitation and Percoll gradient separation. The specific granule fraction (membrane sample containing cytochrome b) and the S2 fraction (cytosol) were collected. The assay mixture contained the membrane sample, EGTA, FAD, MgCl2, GTPγS, and NaN3 in phosphate buffer. Cytosolic factors (S2) were present in excess, and SDS was added as the activator. Superoxide production was monitored by SOD-inhibitable reduction of cytochrome c at 550 nm. B-220 (10 μg/mL) showed no inhibitory effect on either the assembly or the activity of the oxidase. [2]

Human neutrophils were isolated from buffy coats of healthy adults. After dextran sedimentation, hypotonic lysis of erythrocytes, and Hypaque-Ficoll gradient centrifugation, neutrophils were resuspended in Krebs-Ringer phosphate buffer containing glucose, Ca2+, and Mg2+ (KRG, pH 7.3). [2]
Measurement of neutrophil superoxide anion production (extracellular) used an isoluminol-enhanced chemiluminescence system. The reaction mixture (1.0 mL) contained 3×10^5 neutrophils, HRP (4 U), and isoluminol (2×10^-5 M). Cells were preincubated with or without B-220 for 5 min at 37°C, then stimulated with PMA (5×10^-8 M), fMLP (10^-7 M), or serum-opsonized yeast. Light emission was continuously recorded using a luminometer. [2]
Measurement of intracellular superoxide anion production used a luminol-enhanced chemiluminescence system with SOD (20 U/mL) and catalase (2000 U/mL) added to the reaction mixture. The mixture contained cells, luminol (2×10^-5 M), SOD, catalase, and B-220 (5 or 10 μg/mL). After 5 min preincubation at 37°C, cells were stimulated with PMA (5×10^-8 M) or opsonized yeast, and light emission was recorded. fMLP did not induce intracellular ROS production. [2]
Measurement of superoxide anion production using SOD-inhibitable cytochrome c reduction: Neutrophils (2×10^6 cells) were added to a cuvette containing KRG and cytochrome c (1.5 mg in 1 mL final volume). Cells were activated by PMA (5×10^-8 M), and the change in absorbance at 550 nm was followed continuously for 15 min using a double-beam spectrophotometer. [2]
Measurement of hydrogen peroxide production from neutrophils: A fluorometric assay using PHPA was performed. Cells (2×10^6) were added to cuvettes containing HRP and PHPA, then activated by PMA (10^-7 M). Fluorescence was measured at excitation 317 nm and emission 400 nm. [2]
Granule mobilization assay (CR3 surface expression): Neutrophils preincubated with or without B-220 (10 μg/mL) were stimulated with fMLP (10^-7 M) for 5 min, then fixed in 4% paraformaldehyde in PBS for 30 min on ice. Phycoerythrin-conjugated monoclonal antibody against CD11b (10 μL) was added to the cell pellet (approx. 10^6 cells) and incubated for 30 min on ice, then washed twice with PBS. Antibody binding (reflecting CR3 molecules on cell surface) was quantified by flow cytometry. [2]
Granule secretion assay (vitamin B12-binding protein release): After activation of neutrophils with fMLP (10^-7 M) for 5 min, the release of vitamin B12-binding protein (marker of specific granules) was assayed using the cyanocobalamin technique. [2]
Phagocytosis assay: Human neutrophils (10^6 cells/mL) were mixed with serum-opsonized fluorescein-labeled yeast cells at a ratio of 1:5. Cells were allowed to phagocytose yeast particles for 5-10 min at 37°C. The process was terminated by adding an equal volume of ice-cold 4% paraformaldehyde in PBS. Phagocytosis was determined by flow cytometry. B-220 (10 μg/mL) was added before stimulation. [2]

[1]. Pharmaceutical formulation of B220 for topical treatment of herpes. EP 2489354 A1.

[2]. The synthetic non-toxic drug 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline inhibits neutrophil production of reactive oxygen species. J Leukoc Biol. 1999 Jun;65(6):771-7.

B-220 (B220) is described as a non-toxic drug. It has antiviral properties and also possesses anticancer qualities and provides protection against oxidative stress. It is a remarkably efficient DNA triplex stabilizer and interacts with RNA with an affinity similar to that of DNA. [2]
The pharmaceutical composition for topical administration comprises B-220 in a pharmaceutically acceptable carrier, e.g., in an amount of 0.1-10% (w/w), preferably 0.5-5% (w/w), e.g., about 1% (w/w). One embodiment claims 5.5% (w/w). The composition can be in the form of a cream, liquid, lotion, gel, spray, foam, or ointment, and can be contained in a patch, stick, spray dispenser, tube, or pen. Optional additional therapeutically active ingredients include antiviral agents, antibiotics, analgesics, anaesthetic agents, antiphlogistic agents, and anti-inflammatory agents. [1]
An exemplary preparation: B-220 (1 part) mixed with white petrolatum (99 parts) in a homogenizer to provide an ointment free of other components. This ointment is stable for more than 24 months; no stabilizers or conditioners were added. Variants with higher percentage (e.g., 2.5% w/w) can be prepared similarly. [1]
The beneficial effects of B-220 are achieved through inhibition of the phagocyte NADPH oxidase activity, thereby reducing oxidative stress. The drug reduces the capacity of neutrophils to generate reactive oxygen species, both extracellularly and intracellularly. The inhibition is not due to direct radical scavenging. B220 exerts its effects on the signaling activity in cells downstream of PKC. It inhibits fMLP-induced activation of NADPH oxidase to a much higher degree than granule mobilization. [2]

C20H22N4

318.41548

318.184

112228-65-6

130705

Light yellow to yellow solid powder

1.2±0.1 g/cm3

518.8±50.0 °C at 760 mmHg

267.6±30.1 °C

0.0±1.4 mmHg at 25°C

1.650

4.45

0

3

3

24

439

0

FPLSGFJELWCFTH-UHFFFAOYSA-N

InChI=1S/C20H22N4/c1-13-11-16-17(12-14(13)2)22-20-19(21-16)15-7-5-6-8-18(15)24(20)10-9-23(3)4/h5-8,11-12H,9-10H2,1-4H3

2-(2,3-dimethylindolo[3,2-b]quinoxalin-6-yl)-N,N-dimethylethanamine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)